捕食線蟲(chóng)性真菌的分離鑒定與主要生物學(xué)特性的研究
發(fā)布時(shí)間：2018-09-03 08:21
【摘要】：捕食線蟲(chóng)性真菌作為自然界中動(dòng)物或植物寄生線蟲(chóng)的天敵,是世界廣布性的一類(lèi)微小真菌。利用捕食線蟲(chóng)性真菌對(duì)感染性幼蟲(chóng)的生物防治,在控制家畜寄生線蟲(chóng)病的替代措施中是一種理想選擇。鑒于此,本研究目的是調(diào)查與牛羊有關(guān)的不同樣品中捕食線蟲(chóng)性真菌的種類(lèi)和出現(xiàn)頻率,探尋可用于生物防治家畜寄生線蟲(chóng)的潛在菌種；并通過(guò)光學(xué)顯微鏡及掃描電鏡觀察研究鞭式達(dá)丁屯氏菌(Duddingtonia flagrans)與捻轉(zhuǎn)血矛線蟲(chóng)(Haemonchus contortus)感染性幼蟲(chóng)(L3)、自由生活線蟲(chóng)秀麗隱桿線蟲(chóng)(Caenorhabditis elegans)相互作用的動(dòng)態(tài)過(guò)程：也觀察了培養(yǎng)溫度和pH值對(duì)捕食線蟲(chóng)性真菌分離株一長(zhǎng)孢隔指孢菌(Dactylella leptospora)(IS-307)生長(zhǎng)的影響。(1)本研究自2013年4月至2014年12月,從中國(guó)20省(自治區(qū)、直轄市)43個(gè)市、縣89個(gè)采樣點(diǎn),采集樣品共計(jì)1408份,其中糞便1 050份,草場(chǎng)上的土壤108份,糞堆149份及廄舍周?chē)耐寥?01份。從3094個(gè)檢測(cè)平皿中分離到捕食線蟲(chóng)性真菌614株,鑒定為4屬20種,包括節(jié)叢孢屬(Arthrobotrys)13種,其中11個(gè)已定種和2個(gè)未定種；單頂孢屬(Monacrosporium)4種；隔指孢屬(Dactylella)2種；達(dá)丁屯氏屬(Duddingtonia)1種。捕食線蟲(chóng)性真菌在所有樣品中的檢出率為19.82%；在糞便、糞堆、廄舍周?chē)寥�、草�?chǎng)上土壤中的檢出率分別為：11.04%、33.42%、39.48%、52.57%。此次調(diào)查的所有樣品中,主要種類(lèi)為少孢節(jié)叢孢少孢變種(Arthrobotrys oligospora var. oligospora),圓錐節(jié)叢孢(Arthrobotry conoides),指狀節(jié)叢孢(Arthrobotry dactyloides),彎孢節(jié)叢孢(Arthrobotry musiformis),多孢節(jié)叢孢(Arthrobotry superba),奇妙單頂孢(Monacrosporium thaumasiun)和D. flagrans,檢出率分別為：13.06%、0.52%、0.68%、0.61%、1.10%、0.48%、1.20%,其余種類(lèi)的檢出率小于0.4%。在已記錄的這些捕食線蟲(chóng)性真菌中,17種產(chǎn)生黏性網(wǎng),出現(xiàn)頻率為94.46%,僅2種產(chǎn)生其它捕食結(jié)構(gòu),如收縮環(huán)或者黏性球與非收縮環(huán),所占比例為3.74%。A. oligospora是最常見(jiàn)的種,在每個(gè)采樣地區(qū)都己分離到,而A.conoides,A. dactyloides, A. musiformis, A. superba及M. thaumasiun分別在不同地區(qū)出現(xiàn)。D. flagrans是動(dòng)物寄生線蟲(chóng)生物防治中重要的候選菌種,此次調(diào)查中,從寧夏、甘肅、黑龍江、山東、貴州都有檢出,在糞便及糞堆中的檢出率分別為0.87%、4.35%。(2)將D. flagrans培養(yǎng)于0.2g/L玉米粉瓊脂培養(yǎng)基(CAM)上,用于掃描電鏡觀察時(shí)則接種于覆蓋有纖維素膜的CAM上。培養(yǎng)7 d后,加入試驗(yàn)線蟲(chóng),隨之在相互作用后的0、1、2、3、4、6、8、12、16、20、24、36和48 h取含有蟲(chóng)體與真菌的瓊脂塊觀察,當(dāng)蟲(chóng)體剛被真菌捕捉即開(kāi)始計(jì)為0h。結(jié)果表明,該菌在未加入試驗(yàn)線蟲(chóng)培養(yǎng)36 h后可自發(fā)形成捕食結(jié)構(gòu)；在加蟲(chóng)相互作用30min后,可誘導(dǎo)產(chǎn)生捕食器,此時(shí)也有蟲(chóng)體被捕捉；捕捉后4 h,菌絲侵入秀麗隱桿線蟲(chóng)導(dǎo)致其快速死亡,而L3被菌絲侵入致死則需要20-24 h；捕捉后24 h,整個(gè)秀麗隱桿線蟲(chóng)被菌絲侵占并完全消化；而L3則需要36 h被菌絲完全侵占,48 h被完全消化僅留表皮層。(3)在玉米粉瓊脂培養(yǎng)基上,觀察捕食線蟲(chóng)性真菌分離株D. leptospora(IS-307)在4、5、6、7、8、9、10、11、12不同pH值下以及在15℃、20℃、25℃、30℃不同溫度下的生長(zhǎng)情況。結(jié)果顯示,該菌在pH值為4-12,溫度為15℃-30℃之間的環(huán)境下均能生長(zhǎng),最佳培養(yǎng)條件pH值為11.0,溫度為25℃。
[Abstract]:Nematode-trapping fungi, as natural enemies of parasitic nematodes of animals and plants in nature, are a kind of microfungi widely distributed in the world. The biological control of infectious larvae by nematode-trapping fungi is an ideal alternative to control parasitic nematodes of livestock. In view of this, the purpose of this study is to investigate the relationship between cattle and sheep. Species and frequencies of nematode-trapping fungi in different samples were studied to explore potential fungi for biological control of parasitic nematodes in livestock. Infectious larvae of Duddingtonia flagrans and Haemonchus contortus (L3), free-living lines were observed by light microscope and scanning electron microscope. Dynamics of interaction between Caenorhabditis elegans: Effects of culture temperature and pH value on the growth of nematode-trapping fungal isolate Dactylella leptospora (IS-307) were also observed. (1) This study was conducted from April 2013 to December 2014 in 43 cities and counties of 20 provinces (autonomous regions, municipalities directly under the Central Government) in China. A total of 1 408 samples were collected from 1 050 feces, 108 grassland soils, 149 fecal heaps and 101 surrounding stables. 614 strains of nematode-trapping fungi were isolated from 3 094 detection dishes and identified as 20 species belonging to 4 genera, including 13 species of Arthrobotrys, of which 11 were identified and 2 were undetermined. There are 4 species of Monacrosporium, 2 species of Dactylella and 1 species of Duddingtonia. The detection rate of nematode-trapping fungi in all samples is 19.82%. The detection rates of nematode-trapping fungi in feces, manure heaps, soil around stables and grassland soil are 11.04%, 33.42%, 39.48% and 52.57% respectively. Arthrobotrys oligospora var. oligospora, Arthrobotrys conoides, Arthrobotrys dactyloides, Arthrobotrys musiformis, Arthrobotrys superba, Monacrosporium thaumasium and D. flagrans were detected. Among the recorded nematode-trapping fungi, 17 species produced viscous nets with a frequency of 94.46%. Only 2 species produced other predatory structures, such as contractile rings or viscous spheres and non-contractile rings, accounting for 3.74%. A. conoides, A. dactyloides, A. musiformis, A. superba and M. thaumasiun were found in different areas. D. flagrans is an important candidate strain for biological control of animal parasitic nematodes. In this investigation, it was found in Ningxia, Gansu, Heilongjiang, Shandong and Guizhou provinces, and in feces and dunghills. (2) D. flagrans were cultured on 0.2 g/L corn flour agar medium (CAM) and inoculated on CAM covered with cellulose membrane for scanning electron microscopy observation. After 7 days of culture, experimental nematodes were added, and then the agar containing worms and fungi was obtained at 0, 1, 2, 3, 4, 6, 8, 12, 20, 24, 36 and 48 hours after interaction. The results showed that the predatory structure could be formed spontaneously after 36 h incubation without the experimental nematode, the predator could be induced after 30 min interaction with the insect, and the parasite was also caught; 4 h after the capture, the mycelium invaded C. elegans and the L3 was killed quickly. It took 20-24 hours for the mycelium to invade and kill, 24 hours after capture, the whole C. elegans was invaded and digested completely by the mycelium, while L3 was invaded completely by the mycelium for 36 hours and digested completely for 48 hours, leaving only the epidermis. (3) The predatory fungal isolate D. leptospora (IS-307) was observed on corn flour agar medium at 4,5,6,7,8,9,10,11,12 hours. The results showed that the strain could grow at pH 4-12 and temperature 15-30, and the optimum culture conditions were pH 11.0 and temperature 25.
【學(xué)位授予單位】：西北民族大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S855.9
本文編號(hào)：2219421
[Abstract]:Nematode-trapping fungi, as natural enemies of parasitic nematodes of animals and plants in nature, are a kind of microfungi widely distributed in the world. The biological control of infectious larvae by nematode-trapping fungi is an ideal alternative to control parasitic nematodes of livestock. In view of this, the purpose of this study is to investigate the relationship between cattle and sheep. Species and frequencies of nematode-trapping fungi in different samples were studied to explore potential fungi for biological control of parasitic nematodes in livestock. Infectious larvae of Duddingtonia flagrans and Haemonchus contortus (L3), free-living lines were observed by light microscope and scanning electron microscope. Dynamics of interaction between Caenorhabditis elegans: Effects of culture temperature and pH value on the growth of nematode-trapping fungal isolate Dactylella leptospora (IS-307) were also observed. (1) This study was conducted from April 2013 to December 2014 in 43 cities and counties of 20 provinces (autonomous regions, municipalities directly under the Central Government) in China. A total of 1 408 samples were collected from 1 050 feces, 108 grassland soils, 149 fecal heaps and 101 surrounding stables. 614 strains of nematode-trapping fungi were isolated from 3 094 detection dishes and identified as 20 species belonging to 4 genera, including 13 species of Arthrobotrys, of which 11 were identified and 2 were undetermined. There are 4 species of Monacrosporium, 2 species of Dactylella and 1 species of Duddingtonia. The detection rate of nematode-trapping fungi in all samples is 19.82%. The detection rates of nematode-trapping fungi in feces, manure heaps, soil around stables and grassland soil are 11.04%, 33.42%, 39.48% and 52.57% respectively. Arthrobotrys oligospora var. oligospora, Arthrobotrys conoides, Arthrobotrys dactyloides, Arthrobotrys musiformis, Arthrobotrys superba, Monacrosporium thaumasium and D. flagrans were detected. Among the recorded nematode-trapping fungi, 17 species produced viscous nets with a frequency of 94.46%. Only 2 species produced other predatory structures, such as contractile rings or viscous spheres and non-contractile rings, accounting for 3.74%. A. conoides, A. dactyloides, A. musiformis, A. superba and M. thaumasiun were found in different areas. D. flagrans is an important candidate strain for biological control of animal parasitic nematodes. In this investigation, it was found in Ningxia, Gansu, Heilongjiang, Shandong and Guizhou provinces, and in feces and dunghills. (2) D. flagrans were cultured on 0.2 g/L corn flour agar medium (CAM) and inoculated on CAM covered with cellulose membrane for scanning electron microscopy observation. After 7 days of culture, experimental nematodes were added, and then the agar containing worms and fungi was obtained at 0, 1, 2, 3, 4, 6, 8, 12, 20, 24, 36 and 48 hours after interaction. The results showed that the predatory structure could be formed spontaneously after 36 h incubation without the experimental nematode, the predator could be induced after 30 min interaction with the insect, and the parasite was also caught; 4 h after the capture, the mycelium invaded C. elegans and the L3 was killed quickly. It took 20-24 hours for the mycelium to invade and kill, 24 hours after capture, the whole C. elegans was invaded and digested completely by the mycelium, while L3 was invaded completely by the mycelium for 36 hours and digested completely for 48 hours, leaving only the epidermis. (3) The predatory fungal isolate D. leptospora (IS-307) was observed on corn flour agar medium at 4,5,6,7,8,9,10,11,12 hours. The results showed that the strain could grow at pH 4-12 and temperature 15-30, and the optimum culture conditions were pH 11.0 and temperature 25.
【學(xué)位授予單位】：西北民族大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S855.9
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，本文編號(hào)：2219421
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